This invention relates to antibodies.
Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulphide bonds and two light chains, each light chain being linked to a respective heavy chain by disulphide bonds. The general structure of an antibody of class IgG (i.e. an immunoglobulin (Ig) of class gamma (G)) is shown schematically in FIG. 1 of the accompanying drawings.
Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain. The constant domains in the light and heavy chains are not involved directly in binding the antibody to antigen.
The variable domains of each pair of light and heavy chains form the antigen binding site. The domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs) (see reference 11). The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
The CDRs are held in close proximity by the framework regions and, with the CDRs from the other domain, contribute to the formation of the antigen binding site.
According to one aspect of the present invention there is provided an antibody having at least one CDR which is foreign with respect to the constant region of the antibody, said at least one foreign CDR being selected from CDRs substantially as identified in FIG. 2, namely residues 31 to 35 (SEQ ID NO:1), 50 to 65 (SEQ ID NO:2) and 95 to 102 (SEQ ID NO:3), of the heavy chain and residues 24 to 34, 50 to 56 and 89 to 97 of the light chain, the antibody being capable of binding effectively to the antigen Campath-1.
The term xe2x80x9cforeignxe2x80x9d is used in relation to the CDR(s) and constant region to mean of different origin.
In FIG. 2 and elsewhere in the specification amino acid residues are identified by the conventionally used one letter symbols, as follows:
In this specification, effective antibody-antigen binding is used to mean that antibody effects 50% binding to antigen at antibody concentrations of less than or equal to 70 ug/ml, preferably at concentrations of less than or equal to 7 ug/ml. Binding affinity may be tested by assay procedures such as are described in Example 1 herein, eg using Campath-1 antigen obtained from a glycolipid extract from human spleen. (ug=microgram)
Thus, a standard procedure for the extraction of glycolipids can be applied to the extraction of the Campath-1 antigen from human spleens. This standard extraction procedure involves the treatment of 1 volume of homogenised human spleen tissue with 3 volumes of water, 11 volumes of methanol and 5.4 volumes of chloroform. After mixing precipitated material is discarded and a further 3.5 volumes of water are added, followed by further mixing. The mixture is then allowed to separate into two phases, the lower chloroform-containing phase is discarded and the upper aqueous phase is concentrated to provide a crude extract of the Campath-1 antigen, which can if desired be purified further by affinity chromatography, for example using the YTH66.9 antibody referred to hereinafter.
The antibody of the present invention desirably has a light chain with at least one CDR selected from CDRs substantially as identified in FIG. 2 and a heavy chain with at least one CDR selected from CDRs substantially as identified in FIG. 2.
As a further possibility, the antibody of the present invention preferably has three heavy chain CDRs substantially as identified in FIG. 2, or three light chain CDRs substantially as identified in FIG. 2. More preferably, the antibody has all six heavy and light chain CDRs substantially as identified in FIG. 2.
Hence, in a preferred aspect the present invention provides an antibody having heavy and light chain CDRs which are foreign with respect to the constant region of the antibody, said CDRs being substantially as identified in FIG. 2, namely residues 31 to 35, 50 to 65 and 95 to 102 of the heavy chain and residues 24 to 34, 50 to 56 and 89 to 97 of the light chain, the antibody being capable of binding effectively to the antigen Campath-1.
The CDRs identified in FIG. 2 are of rat origin and may be combined with a range of different variable domain framework regions, as desired, including, eg, framework regions of rat or human origin.
In a further aspect the present invention provides an antibody having heavy and light chain variable domains as identified in the lower lines of sequence information in FIG. 2, namely residues 1 to 113 (SEQ ID NO:7) of the heavy chain and residues 1 to 108 (SEQ ID NO:8) of the light chain, the CDRs and constant region of the antibody being foreign with respect to one another, the antibody being capable of binding effectively to the antigen Campath-1.
Such an antibody comprises CDRs and framework regions of rat origin.
The invention also provides an antibody having heavy and light chain variable domains as identified in the upper lines of sequence information in FIG. 2, namely residues 1 to 113 (SEQ ID NO:9) of the heavy chain and residues 1 to 108 (SEQ ID NO:10) of the light chain, and that will bind effectively to the antigen Campath-1.
Such an antibody comprises CDRs of rat origin in framework regions of human origin.
Such an antibody may be modified by having a phenylalanine group at residue 27 of the heavy chain in place of serine, and possibly also by having a threonine group at residue 30 of the heavy chain in place of serine. A Ser(27) to Phe mutation is found to increase antibody-antigen binding significantly. However, the mutation of Ser (30) to Thr (in the human framework with the Ser (27) to Phe mutation) has little effect on binding affinity. This illustrates that point mutations in the antibody may have a major effect or little effect on the affinity of the antibody for the antigen. Although the two changes Ser (27) to Phe and Ser (30) to Thr are located within the framework region as defined in reference 11, they lie within the hypervariable loop Hl as defined in reference 18. It is accordingly believed that some changes in the CDRs may similarly be made without necessarily having an adverse effect on antibody-antigen affinity. References to CDRs substantially as identified in FIG. 2 are accordingly intended to include within their scope not only CDRs identical to those identified in FIG. 2 but also variants of such CDRs, subject to the requirement of the antibody binding effectively to Campath-1.
The antibody is preferably in biologically pure form, desirably being at least 95% (by wt) free of other biological materials.
The remainder of the antibody, namely the heavy and light chain constant domains and possibly also variable domain framework regions and one or more CDRs, can be based on antibodies of various different types as desired including, eg, rat and human antibodies of different classes. Thus, the constant domains can be selected to have desired effector functions appropriate to the intended use of the antibody. For example, for therapeutic purposes, human IgG1 and rat IgG2b are currently favoured isotypes. Further, of the human IgG isotypes, IgG1 and IgG3 appear to be the most effective for complement and cell mediated lysis, and therefore for killing tumour cells. For other purposes other isotypes may be favoured, eg, rat IgM, IgG1, IgG2a, IgG2c, human IgG2, IgG4 etc. For human therapy it is particularly desirable to use human isotypes, to minimise antiglobulin responses during therapy.
The Campath-1 antigen is strongly expressed on virtually all human lymphocytes and monocytes, but is absent from other blood cells including the hemopoietic stem cells, the antigen being described by Hale et al in Blood, 1983, 62, 873-882 (reference 6). That paper describes the antibody YTH66.9 which is specific for the Campath-1 antigen, this antibody being available from Serotec of 22 Bankside, Station Approach,. Kidlington, Oxford, England, under the designation YTH 66.9 HL. A further series of antibodies to Campath-1 have been produced, including rat monoclonal antibodies of IgM, IgG2a, and IgG2c isotypes (reference 7) and more recently IgG1 and IgG2b isotypes have been isolated as class switch variants from the IgG2a secreting cell line YTH 34.5HL (reference 8). All of these antibodies with the exception of the rat IgG2c isotype are able to efficiently lyse human lymphocytes with human complement.
In addition, the IgG2b antibody YTH 34.5HL-G2b, but not the other isotypes, is effective in antibody dependent cell mediated cytotoxicity (ADCC) with human effector cells (reference 8). These rat monoclonal antibodies have found important application in the context of immunosuppression, for control of graft-versus-host disease in bone marrow transplantation (reference 6); the management of organ rejection (reference 9); the prevention of marrow rejection and in the treatment of various lymphoid malignancies (reference 10). For in-vivo use, the IgG2b antibody YTH 34.5HL-G2b seems to be the most effective at depleting lymphocytes, but the use of any of the antibodies in this group is limited by the antiglobulin response which can occur within two weeks of the initiation of treatment (reference 10).
Antibodies in accordance with the invention, particularly those based on human isotypes, thus have good therapeutic potential. In particular, the availability of a reshaped human antibody with specificity for the Campath-1 antigen should permit a full analysis of the in vivo potency and immunogenicity of an anti-lymphocyte antibody with wide therapeutic potential. Such reshaped antibodies have been used in the treatment of patients with non-Hodgkin lymphoma, as well as in the treatment of some cases of autoimmune disease. Further trials with organ graft patients, particularly kidney graft patients, are proposed. Even if anti-idiotypic responses are eventually observed, considerable therapeutic benefit could be derived by an extended course of treatment. In addition an antiglobulin response restricted to idiotype should be circumvented by using a series of antibodies with different idiotype (reference 20). In principle, the idiotype of the reshaped Campath-1 could be changed by altering the hypervariable regions or the framework regions: evidence from a reshaped antibody specific for the hapten nitrophenyl acetate suggests that the recognition by anti-idiotypic antisera and anti-idiotypic monoclonal antibodies is influenced by residues in the framework region (reference 5). Thus recycling the hypervariable regions on different human framework regions should change the idiotype, although ultimately it could focus the response directly onto the binding site for Campath-1 antigen. Although such focussing would be undesirable for Campath-1 antibodies, it could be an advantage for the development of anti-idiotypic vaccines.
In a further aspect, the invention thus provides a method of treating patients, particularly humans, with cancers, particularly lymphomas, or for immunosuppression purposes, comprising administering antibodies in accordance with the invention.
Antibodies in accordance with the present invention may be formulated for administration to patients by mixing antibody purified in conventional manner with a physiologically acceptable diluent or carrier, possibly in admixture with other therapeutic agents such as other antibodies. In one example, purified antibody was reconstituted in a commercially available human plasma protein solution (minus gamma globulin). The formulation was administered by intravenous infusion at the rate of 5 mg antibody per day for at least 10 days.
Antibodies in accordance with the invention can be produced in various different ways, as will be described in greater detail in the Examples following.
Heavy and light chain variable domains are conveniently produced separately and assembled with the remainder of an antibody of desired origin, eg desired human isotype.
Genes encoding the variable domains of an antibody of desired structure may be produced, and attached to genes encoding the constant domains of an antibody of desired isotype. Genes encoding variable domains can be derived from hybridoma cDNA or from the chromosome. Genes encoding the variable domains are also conveniently constructed by gene synthesis techniques or by site directed mutagenesis using long synthetic oligonucleotides. Expression is conveniently effected by transforming a cell line, eg an immortalised mammalian cell line such as a myeloma cell line, with expression vectors including DNA coding for the variable domains and for the remainder of the antibody and culturing the transfomed cell line to produce the desired antibody.
In another aspect the invention provides a process for the preparation of an antibody having at least one CDR (complementarity determining region) which is foreign with respect of the constant region of the antibody, said at least one foreign CDR being selected from CDRs substantially as identified in FIG. 2, that is amino acid residues 31 to 35, 50 to 65 and 95 to 102 of the heavy chain as shown in FIG. 2a, and amino acid residues 24 to 34, 50 to 56 and 89 to 97 of the light chain as shown in FIG. 2b, the antibody being capable of binding effectively to the antigen Campath-1, which process comprises culturing a cell capable of expressing the antibody in order to effect express-ion thereof.
It will be appreciated that the antibody may be used in a form which retains the CDRs but lacks other parts of the whole molecule not essential to its binding efficacy, in particular a F(abxe2x80x2)2 fragment, and the word antibody is used herein to include such materials.